Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

Sensory neuronal STAT3 is critical for IL-31 receptor expression and inflammatory itch.

IL-31 receptor blockade suppresses pruritus of atopic dermatitis. However, cell-type-specific contributions of IL-31 receptor to itch, its expression mechanism, and the downstream signaling pathway to induce itch remain unknown. Here, using conditional knockout mice, we demonstrate that IL-31-induced itch requires sensory neuronal IL-31 receptor and STAT3. We find that IL-31 receptor expression is dependent on STAT3 in sensory neurons. In addition, pharmacological experiments suggest that STAT3 activation is important for the itch-inducing signaling downstream of the IL-31 receptor. A cutaneous IL-31 injection induces the nuclear accumulation of activated STAT3 first in sensory neurons that abundantly express IL-31 receptor and then in other itch-transmitting neurons. IL-31 enhances itch induced by various pruritogens including even chloroquine. Finally, pruritus associated with dermatitis is partially dependent on sensory neuronal IL-31 receptor and strongly on sensory neuronal STAT3. Thus, sensory neuronal STAT3 is essential for IL-31-induced itch and further contributes to IL-31-independent inflammatory itch.

SETBP1 is dispensable for normal and malignant hematopoiesis.

SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1fl/fl. Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.

VCre/VloxP and SCre/SloxP as Reliable Site-Specific Recombination Systems for Genome Engineering.

The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved.

Polycomb repressive complexes 1 and 2 are each essential for maintenance of X inactivation in extra-embryonic lineages.

In female mammals, one of the two X chromosomes becomes inactivated during development by X-chromosome inactivation (XCI). Although Polycomb repressive complex (PRC) 1 and PRC2 have both been implicated in gene silencing, their exact roles in XCI during in vivo development have remained elusive. To this end, we have studied mouse embryos lacking either PRC1 or PRC2. Here we demonstrate that the loss of either PRC has a substantial impact on maintenance of gene silencing on the inactive X chromosome (Xi) in extra-embryonic tissues, with overlapping yet different genes affected, indicating potentially independent roles of the two complexes. Importantly, a lack of PRC1 does not affect PRC2/H3K27me3 accumulation and a lack of PRC2 does not impact PRC1/H2AK119ub1 accumulation on the Xi. Thus PRC1 and PRC2 contribute independently to the maintenance of XCI in early post-implantation extra-embryonic lineages, revealing that both Polycomb complexes can be directly involved and differently deployed in XCI.

PCGF1-PRC1 links chromatin repression with DNA replication during hematopoietic cell lineage commitment.

Polycomb group proteins (PcG), polycomb repressive complexes 1 and 2 (PRC1 and 2), repress lineage inappropriate genes during development to maintain proper cellular identities. It has been recognized that PRC1 localizes at the replication fork, however, the precise functions of PRC1 during DNA replication are elusive. Here, we reveal that a variant PRC1 containing PCGF1 (PCGF1-PRC1) prevents overloading of activators and chromatin remodeling factors on nascent DNA and thereby mediates proper deposition of nucleosomes and correct downstream chromatin configurations in hematopoietic stem and progenitor cells (HSPCs). This function of PCGF1-PRC1 in turn facilitates PRC2-mediated repression of target genes such as Hmga2 and restricts premature myeloid differentiation. PCGF1-PRC1, therefore, maintains the differentiation potential of HSPCs by linking proper nucleosome configuration at the replication fork with PcG-mediated gene silencing to ensure life-long hematopoiesis.

A case of male perineal aggressive angiomyxoma with expressions of female hormone receptors.

Introduction: Aggressive angiomyxoma is a rare mesenchymal tumor in females of reproductive age that occurs in the pelvis and perineal zone with a high risk of local infiltration and recurrence. Male aggressive angiomyxoma in perineal zone is very rare. Case presentation: A 63-year old male presented to our hospital with chief complaint of perineal mass. He was diagnosed with perineal lipoma by needle biopsy 3 years before. Computed tomography revealed a 16 cm perineal tumor without lymph node or distal metastasis. As the tumor had gradually increased, we performed tumor resection. The histological diagnosis of tumor using immunohistochemistry was aggressive angiomyxoma with complete resection. Moreover, expressions of both estrogen and progesterone receptors were recognized. No recurrence was seen 9 months after surgery. Conclusion: Male aggressive angiomyxoma is a very rare tumor, which has expressions of female hormone receptors. Hormonal therapy might be effective for perioperative therapy or recurrence.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells.

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes.

Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.

The Influence of Young Age on Difficulties in the Surgical Resection of Carotid Body Tumors.

This study evaluated patient characteristics that affect the complexity and difficulties of performing surgery to resect carotid body tumors (CBTs). We retrospectively reviewed the medical records of 20 patients with 21 CBTs who were enrolled in the study. The median patient age was 46 years and the mean tumor diameter was 37.6 mm. The mean blood loss and operative time were 40.3 mL and 183 min, respectively. Four patients underwent resection of the carotid artery followed by reconstruction. These four patients were between 18 to 23 years of age at the time of surgery. The mean blood loss and operative time in these patients were 166 mL and 394 min, respectively, which differed significantly from those of older patients. Therefore, young age influenced the difficulties faced in surgical resection of CBT, with an increased risk of blood loss and carotid artery resection.

Genetic alterations in squamous cell lung cancer associated with idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) are at higher risk of developing lung cancers including squamous cell lung carcinoma (SCC), which typically carries a poor prognosis. Although the molecular basis of cancer development subsequent to IPF has not been fully investigated, we recently reported two epigenetic phenotypes characterized by frequent and infrequent DNA hypermethylation in SCC, and an association of the infrequent hypermethylation phenotype with IPF-associated SCCs. Here, we conducted targeted exon sequencing in SCCs with and without IPF using the Human Lung Cancer Panel to investigate the genetic basis of IPF-associated SCC. SCCs with and without IPF displayed comparable numbers of total mutations (137 ± 22 vs 131 ± 27, P = .5), nonsynonymous mutations (72 ± 14 vs 69 ± 16, P = .5), indels (3.0 ± 3.5 vs 3.0 ± 3.9, P = 1) and synonymous mutations (62 ± 9.1 vs 60 ± 12, P = .5). Signature 1 was the predominant signature in SCCs with and without IPF. SETD2 and NFE2L2 mutations were significantly associated with IPF (44% vs 13%, P = .03 for SETD2; 38% vs 10%, P = .04 for NFE2L2). MYC amplification, assessed by copy number variant analysis, was also significantly associated with IPF (18.8% vs 0%, P = .04). Mutations in TP53 and CDKN2A were observed relatively frequently in SCCs with frequent hypermethylation (P = .02 for TP53 and P = .06 for CDKN2A). Survival analysis revealed that the SETD2 mutation was significantly associated with worse prognosis (P = .04). Collectively, we found frequent involvement of SETD2 and NFE2L2 mutations and MYC amplification in SCCs with IPF, and an association of a SETD2 mutation with poorer prognosis.

Clinical phenotypes and genetic analyses for diagnosis of systemic autoinflammatory diseases in adult patients with unexplained fever.

OBJECTIVE: To make an accurate diagnosis of systemic autoinflammatory diseases (SAIDs), clinical and genetic analyses were performed in patients with unexplained fever. METHODS: The clinical phenotype and genomic variants of 11 genes responsible for SAIDs were analyzed in 179 Japanese patients with unexplained fever. Genetic analysis was performed by next generation sequencing (NGS) on exons including exon-intron boundaries. RESULTS: Three cases met the diagnostic criteria for SAIDs other than familial Mediterranean fever (FMF). Considering 176 patients with unexplained fever, 43 cases (24.0%) were clinically diagnosed as FMF. Gene variants were found in 53 cases (30.1%) when searching for variants in the 10 disease genes other than the MEFV gene. Among them, the most frequently-identified genes were NLRP3, NOD2, NLRP12, NLRC4, and PLCG2, which accounted for 14, 7, 17, 7, and 6 cases, respectively. These variants were less than 1% of healthy individuals or novel variants, but not regarded as pathogenic since the patients did not meet the diagnostic criteria of SAIDs caused by their identified variants clinically. CONCLUSION: Twenty four percent of Japanese patients with unexplained fever were clinically diagnosed as FMF in this study. Low frequency but not pathogenic variants in genes other than MEFV were identified in 30.1% of the cases. It is not clear how much these gene variants contribute to the inflammatory phenotypes; therefore, further analysis would uncover their autoinflammatory phenotypes that cause fever.

Publisher Correction: Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.

Coagulopathy-related soft tissue hematoma: a comparison between computed tomography findings and clinical severity.

BACKGROUND: Despite increases in the incidence of coagulopathy-related soft-tissue hematoma (CRSH), the relationship between computed tomography (CT) features and clinical severity remains unclear. PURPOSE: To retrospectively evaluate the correlation between CT findings and clinical outcomes in CRSH. MATERIAL AND METHODS: We retrospectively reviewed data of patients diagnosed with CRSH between March 2011 and March 2018. CRSH was morphologically classified according to the presence or absence of the fluid level pattern and was also divided into groups with or without extravasation as per CT findings. These CT findings were compared with the patients' vital signs and laboratory investigation results. RESULTS: A total of 47 patients with CRSH were examined. Fluid level and non-fluid level patterns were observed in 28 (60%) and 19 (40%) patients, respectively. Anticoagulant therapy and extravasation were significantly correlated with the fluid level pattern. However, other clinicolaboratory outcomes, including shock index, hemoglobin, hematocrit, platelet count, and coagulation factors, showed no significant difference between the two patterns. In the comparison of hematomas with and without extravasation, none of the clinicolaboratory outcomes except for anticoagulant therapy showed significant differences. CONCLUSION: CRSH with a fluid level pattern is significantly associated with extravasation. However, extravasation, which is generally suggestive of active bleeding, does not seem to be related to clinical severity in CRSH.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Effects of BMP7 produced by group 2 innate lymphoid cells on adipogenesis.

Group 2 innate lymphoid cells (ILC2s) are type 2 cytokine-producing cells that have important roles in helminth infection and allergic inflammation. ILC2s are tissue-resident cells, and their phenotypes and roles are regulated by tissue-specific environmental factors. While the role of ILC2s in the lung, intestine and bone marrow has been elucidated in many studies, their role in adipose tissues is still unclear. Here, we report on the role of ILC2-derived bone morphogenetic protein 7 (BMP7) in adipocyte differentiation and lipid accumulation. Co-culture of fat-derived ILC2s with pluripotent mesenchymal C3H10T1/2 cells and committed white preadipocyte 3T3-L1 cells resulted in their differentiation to adipocytes and induced lipid accumulation. Co-culture experiments using BMP7-deficient ILC2s revealed that BMP7, produced by ILC2s, induces differentiation into brown adipocytes. Our results demonstrate that BMP7, produced by ILC2s, affects adipocyte differentiation, particularly in brown adipocytes.

Prognostic factors for survival of herpes simplex virus-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) occurs in neonates with disseminated infection of herpes simplex virus (HSV). Little has been reported on the control of rapid HLH progression. We studied the cytokine profile and genetic basis of two index cases with divergent outcomes after early treatment of type 2 HSV infection. One survivor had fever and elevated serum levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), interferon (IFN)-ß, and IFN-γ at diagnosis. The other neonate had no fever or TNF-α production, but significant IL-6 or IFN responses during the treatment course, and died 19 days after birth. Among 16 reported cases of neonatal HSV-HLH including index cases, eight deceased neonates experienced significantly less fever at presentation (p = 0.028), lower platelet counts (p = 0.019), and lower ratios of soluble IL-2 receptor (sIL-2R) to ferritin levels (p = 0.044) than eight survivors. The 100-day overall survival rates were significantly higher in patients with fever (p = 0.004), > 100 × 109/L of platelet counts (p = 0.035) or > 20 of sIL-2R/ferritin ratio at diagnosis (p = 0.004). The first febrile and cytokine responses to HSV infection predict the early outcome of neonatal HSV-HLH.

Human gain-of-function STAT1 mutation disturbs IL-17 immunity in mice.

Gain-of-function (GOF) mutations in the gene for signal transducer and activator of transcription 1 (STAT1) account for approximately one-half of patients with chronic mucocutaneous candidiasis (CMC) disease. Patients with GOF-STAT1 mutations display a broad variety of infectious and autoimmune manifestations in addition to CMC, and those with severe infections and/or autoimmunity have a poor prognosis. The establishment of safe and effective treatments based on a precise understanding of the molecular mechanisms of this disorder is required to improve patient care. To tackle this problem, we introduced the human R274Q GOF mutation into mice [GOF-Stat1 knock-in (GOF-Stat1R274Q)]. To investigate the immune responses, we focused on the small intestine (SI), which contains abundant Th17 cells. Stat1R274Q/R274Q mice showed excess phosphorylation of STAT1 in CD4+ T cells upon IFN-γ stimulation, consistent with the human phenotype in patients with the R274Q mutation. We identified two subpopulations of CD4+ T cells, those with 'normal' or 'high' level of basal STAT1 protein in Stat1R274Q/R274Q mice. Upon IFN-γ stimulation, the 'normal' level CD4+ T cells were more efficiently phosphorylated than those from WT mice, whereas the 'high' level CD4+ T cells were not, suggesting that the level of STAT1 protein does not directly correlate with the level of pSTAT1 in the SI. Inoculation of Stat1R274Q/R274Q mice with Candida albicans elicited decreased IL-17-producing CD4+RORγt+ cells. Stat1R274Q/R274Q mice also excreted larger amounts of C. albicans DNA in their feces than control mice. Under these conditions, there was up-regulation of T-bet in CD4+ T cells. GOF-Stat1R274Q mice thus should be a valuable model for functional analysis of this disorder.

Feasibility of thin-slice abdominal CT in overweight patients using a vendor neutral image-based denoising algorithm: Assessment of image noise, contrast, and quality.

The purpose of this study was to investigate whether the novel image-based noise reduction software (NRS) improves image quality, and to assess the feasibility of using this software in combination with hybrid iterative reconstruction (IR) in image quality on thin-slice abdominal CT. In this retrospective study, 54 patients who underwent dynamic liver CT between April and July 2017 and had a body mass index higher than 25 kg/m2 were included. Three image sets of each patient were reconstructed as follows: hybrid IR images with 1-mm slice thickness (group A), hybrid IR images with 5-mm slice thickness (group B), and hybrid IR images with 1-mm slice thickness denoised using NRS (group C). The mean image noise and contrast-to-noise ratio relative to the muscle of the aorta and liver were assessed. Subjective image quality was evaluated by two radiologists for sharpness, noise, contrast, and overall quality using 5-point scales. The mean image noise was significantly lower in group C than in group A (p < 0.01), but no significant difference was observed between groups B and C. The contrast-to-noise ratio was significantly higher in group C than in group A (p < 0.01 and p = 0.01, respectively). Subjective image quality was also significantly higher in group C than in group A (p < 0.01), in terms of noise and overall quality, but not in terms of sharpness and contrast (p = 0.65 and 0.07, respectively). The contrast of images in group C was greater than that in group A, but this difference was not significant. Compared with hybrid IR alone, the novel NRS combined with a hybrid IR could result in significant noise reduction without sacrificing image quality on CT. This combined approach will likely be particularly useful for thin-slice abdominal CT examinations of overweight patients.